1. Volume 143, Issue 6, Pages 1609-1619.e4 (December 2012)
Liver Failure After Extended Hepatectomy in Mice Is Mediated by a p21-Dependent Barrier to Liver Regeneration 
Kuno Lehmann, Christoph Tschuor, Andreas Rickenbacher, Jae–Hwi Jang, Christian E. Oberkofler, Oliver Tschopp, Simon M. Schultze, Dimitri A. Raptis, Achim Weber, Rolf Graf, Bostjan Humar, Pierre–Alain Clavien 
Gastroenterology 
Volume 143, Issue 6, Pages e4 (December 2012)
DOI: /j.gastro
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2. Figure 1
Extended hepatectomy induces features of liver failure in mice. (A) Modified surgical procedure for the radical resection of mouse liver. Segmental portal vein branches are ligated separately before parenchymal transection. (B) Ischemia of the ligated anterior right liver lobe before parenchymal resection. (C) Serum bilirubin levels at 24 to 168 hours after resection. (D) Plasma alkaline phosphatase (ALKP) levels at 24, 48, and 168 hours after resection. Note the similar patterns for bilirubin and ALKP levels. (E) Serum albumin levels at 48 and 168 hours after resection. (F) Kaplan–Meier estimates for the survival of mice after standard (pHx) or the modified extended (eHx) resection.
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3. Figure 2
Absence of excessive parenchymal injury after extended hepatectomy. (A) Serum alanine aminotransferase (ALT) and high-mobility group protein 1 (Hmgb1) levels at 48 hours after resection. Note the absolute levels of ALT. Necrotic cell death leads to an increase of both serum ALT and Hmgb1 level. (B) Formalin-fixed, paraffin-embedded liver sections 48 hours after resection stained with H&E, against F4/80, Mpo, for DNA fragmentation (TUNEL), and the sinusoidal markers Vegfr3 and CD31. Note the absence of histologically evident tissue injury. (C) Intrahepatic ATP and MDA levels at 48 hours after resection.
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4. Figure 3
LW/BW ratio during the first week after standard and extended resection. (A) The LW/BW indicates a markedly suppressed liver weight gain between 48 and 96 hours after extended hepatectomy. (B) Body weight loss was stronger after eHx than pHx, increasing the LW/BW in eHx mice.
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5. Figure 4
Proliferation markers and DNA ploidy in liver remnants after resection. (A) The percentage of Ki67- and (B) PCNA-positive cells after resection. (C) Liver sections at 48, 72, and 96 hours after resection stained for pH3. Note the prominent, bold nuclei in pH3 stains of pHx mice at 48 hours. (D) Number of hepatocytes undergoing mitosis per high-power field (HPF). (E) Time course of DNA ploidy changes after resection. 2 N, G1-phase cells; 2–4 N, S-phase cells; ≥4 N, G2/M-phase cells. Error bars refer to ± standard deviation. Individual ploidy graphs are shown in Supplemental Figure 1.
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6. Figure 5
Expression of cell-cycle–associated molecules in liver of hepatectomized mice. (A) Quantitative polymerase chain reaction for the expression of the cyclin genes Ccnd1, Ccne1, Ccna2, and Ccnb2. (B) Inhibition of Cdk1 activity as assessed by the ratio of p-Cdk1 vs total Cdk1 protein levels derived from immunoblots 48 hours after resection. (C) Quantitative polymerase chain reaction for the expression of Foxm1b and its downstream targets Skp2 and Csk1b. (D) Quantitative polymerase chain reaction for p21 gene expression (mRNA) and densitometric analysis of p21 total and phosphoprotein immunoblots (protein). Total protein levels were normalized to β-tubulin, and phosphoprotein levels were normalized to total protein. Fold induction was calculated relative to p21 levels in sham-operated animals (see Supplementary Figure 4).
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7. Figure 6
Liver regeneration and survival in wild-type and p21−/− mice after extended hepatectomy. (A) pH3 staining of liver remnants at different time points after extended resection. Note the prominent, bold, pH3-positive nuclei in p21−/− mice as seen in wild-type mice after standard resection. (B) Number of mitoses per high-power field (HPF) and the percentage of Ki67-/PCNA-positive hepatocytes after extended resection in wild-type and p21−/− mice. (C) Gain in liver weight after extended resection. Note the accelerated gain starting in p21−/− mice at 48 hours after eHx. (D) Kaplan–Meier estimates for the survival of wild-type and p21−/− mice after extended resection.
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8. Supplementary Figure 1
Hepatic DNA ploidy profiles after pHx and eHx. Bar charts showing the percentage of hepatic cells with a 2 N, 2–4 N, and ≥4 N DNA content at different times after standard (68%) or extensive resection (86%). The right bars show the percentage of cells with a tetraploid DNA content (4 N) or higher (>4 N). P values indicate significant differences in DNA content distribution between eHx and pHx animals. A minimum of 10,000 cells were analyzed per sample. All cells were included into analysis apart from cells with a <2 N DNA content. The 2 N, 2–4 N, and >4 N populations had a median peak intensity of 2.7×104, 5.5×104, and 11×104, respectively. Three to 5 animals/group were analyzed. Error bars show ± standard deviation.
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9. Supplementary Figure 2
Expression of early promoters of liver regeneration after pHx and eHx. The expression of Il6, Myc, Tnfa, and Hgf was assessed by quantitative polymerase chain reaction (PCR). Error bars indicate ± standard deviation. The activity of c-Jun and Akt proteins at 8 hours after resection was assessed by immunoblots using phospho-specific antibodies. Five mice per group were used. Note the pc-Jun expression tended to be increased in eHx animals.
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10. Supplementary Figure 3
Expression of cell-cycle inhibitors after hepatectomy. The expression of p16, p19, and p27 was assessed by quantitative polymerase chain reaction (PCR). Error bars show ± standard deviation. For p27, expression also is shown on immunohistochemistry and by immunoblots (5 mice/group) at 48 hours after resection. The p27 control shows positive p27 expression in differentiated intestinal cells.
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11. Supplementary Figure 4
p21 total and phosphoprotein levels after standard (68%) and extended (86%) resection. Immunoblot bands show representative levels of p21 protein and its phosphorylation pattern at various times after pHx and eHx as determined from 4–6 animals/time point. S, bands from sham-operated animals. The lower panels show p21 immunofluorescence on paraffin-embedded, formalin-fixed sections from liver. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Note the nuclear localization of p21 in eHx mice.
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12. Supplementary Figure 5
Cholestatic and liver function markers in p21−/− mice after extended hepatectomy. Serum albumin and bilirubin levels at 48 hours after eHx in p21−/− mice. Error bars show ± standard deviation.
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